Strand displacement reaction enables the construction of enzyme-free DNA reaction networks,thus has been widely applied to DNA circuit and nanotechnology.It has the characteristics of high efficiency,universality and ...Strand displacement reaction enables the construction of enzyme-free DNA reaction networks,thus has been widely applied to DNA circuit and nanotechnology.It has the characteristics of high efficiency,universality and regulatability.However,the existing regulation tools cannot enable effective control of the reaction sequence,which undoubtedly limits the construction of complex nucleic acid circuits.Herein,we developed a regulation tool,toehold lock,and achieved strict control of reaction sequence without loss of the main reaction signal output.Furthermore,we applied the tool to scenarios such as seesaw circuits,AND/OR logic gates,and entropy-driven circuits,and respectively demonstrated its significant superiority compared to the original method.We believe that the proposed toehold lock has greatly optimized the efficiency of DNA strand displacement-based networks,and we anticipate that the tool will be widely used in multiple fields.展开更多
Nucleic acid sensing is a 3 decades old but still challenging area of application for different biological sub-domains,from pathogen detection to single cell transcriptomics analysis.The many applications of nucleic a...Nucleic acid sensing is a 3 decades old but still challenging area of application for different biological sub-domains,from pathogen detection to single cell transcriptomics analysis.The many applications of nucleic acid detection and identification are mostly carried out by PCR techniques,sequencing,and their derivatives used at large scale.However,these methods’limitations on speed,cost,complexity and specificity have motivated the development of innovative detection methods among which nucleic acid biosensing technologies seem promising.Toehold switches are a particular class of RNA sensing devices relying on a conformational switch of secondary structure induced by the pairing of the detected trigger RNA with a de novo designed synthetic sensing mRNA molecule.Here we describe a streamlined methodology enabling the development of such a sensor for the RNA-mediated detection of an endangered plant species in a cell-free reaction system.We applied this methodology to help identify the rosewood Dalbergia maritima,a highly trafficked wood,whose protection is limited by the capacity of the authorities to distinguish protected logs from other unprotected but related species.The streamlined pipeline presented in this work is a versatile framework enabling cheap and rapid development of new sensors for custom RNA detection.展开更多
Background： The DNA strand displacement reaction, which uses flexible and programmable DNA molecules as reaction components, is the basis of dynamic DNA nanotechnology, and has been widely used in the design of compl...Background： The DNA strand displacement reaction, which uses flexible and programmable DNA molecules as reaction components, is the basis of dynamic DNA nanotechnology, and has been widely used in the design of complex autonomous behaviors. Results： In this review, we first briefly introduce the concept of toehold-mediated strand displacement reaction and its kinetics regulation in pure solution. Thereafter, we review the recent progresses in DNA complex circuit, the assembly of AuNPs driven by DNA molecular machines, and the detection of single nucleotide polymorphism （SNP） using DNA toehold exchange probes in pure solution and in interface state. Lastly, the applications of toehold-mediated strand displacement in the genetic regulation and silencing through combining gene circuit with RNA interference systems are reviewed. Conclusions： The toehold-mediated strand displacement reaction makes DNA an excellent material for the fabrication of molecular machines and complex circuit, and may potentially be used in the disease diagnosis and the regulation of gene silencing in the near future.展开更多
Cervical cancer is a global public health subject as it affects women in the reproductive ages,and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50%mortality rate.Relativel...Cervical cancer is a global public health subject as it affects women in the reproductive ages,and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50%mortality rate.Relatively scant awareness and limited access to effective diagnosis have led to this enormous disease burden,calling for point-of-care,minimally invasive diagnosis methods.Here,an end-to-end quantitative unified pipeline for diagnosis has been developed,beginning with identification of optimal biomarkers,concurrent design of toehold switch sensors,and finally simulation of the designed diagnostic circuits to assess performance.Using miRNA expression data in the public domain,we identified miR-21-5p and miR-20a-5p as blood-based miRNA biomarkers specific to early-stage cervical cancer employing a multi-tier algorithmic screening.Synthetic riboregulators called toehold switches specific to the biomarker panel were then designed.To predict the dynamic range of toehold switches for use in genetic circuits as biosensors,we used a generic grammar of these switches,and built a neural network model of dynamic range using thermodynamic features derived from mRNA secondary structure and interaction.Second-generation toehold switches were used to overcome the design challenges associated with miRNA biomarkers.The resultant model yielded an adj.R^(2)~0.71,outperforming earlier models of toehold-switch dynamic range.Reaction kinetics modelling was performed to predict the sensitivity of the second-generation toehold switches to the miRNA biomarkers.Simulations showed a linear response between 10 nM and 100 nM before saturation.Our study demonstrates an end-to-end computational workflow for the efficient design of genetic circuits geared towards the effective detection of unique genomic/nucleic-acid signatures.The approach has the potential to replace iterative experimental trial and error,and focus time,money,and efforts.All software including the toehold grammar parser,neural network model and reaction kinetics simulation are available as open-source software(https://github.com/SASTRA-iGEM2019)under GNU GPLv3 licence.展开更多
We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer ...We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer and result in the structure switching of the DNA assembly probes to imitate the target ATP molecule recycling cycles through the toehold-mediated strand displacement reaction,which causes the formation of many dsDNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The organic dye,malachite green,then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals,which can sensitively detect ATP within a dynamic range from 10 to 500 nM in a label-free way.The sensing approach shows a detection limit of 7.3 nM and also has an excellent selectivity for ATP analogue molecules.In addition,this method can detect ATP molecules in diluted human serum samples sensitively,which proves the promising potential to diagnose ATP-related diseases.展开更多
基金the financial support from the National Key Research and Development Program of China(No.2021YFC2701402)the Open Research Fund of State Key Laboratory of Bioelectronics,Southeast University(No.Sklb2021-k06)+2 种基金the Open Foundation of NHC Key Laboratory of Birth Defect for Research and Prevention(Hunan Provincial Maternal and Child Health Care Hospital)(No.KF2020007)Hunan Province Assisted Reproduction and Regenerative Medicine Clinical Demonstration Center Funded Project(No.2020SK4019)the Open Foundation of Translational Medicine National Science and Technology Infrastructure(Shanghai)(No.TMSK-2021-141)。
文摘Strand displacement reaction enables the construction of enzyme-free DNA reaction networks,thus has been widely applied to DNA circuit and nanotechnology.It has the characteristics of high efficiency,universality and regulatability.However,the existing regulation tools cannot enable effective control of the reaction sequence,which undoubtedly limits the construction of complex nucleic acid circuits.Herein,we developed a regulation tool,toehold lock,and achieved strict control of reaction sequence without loss of the main reaction signal output.Furthermore,we applied the tool to scenarios such as seesaw circuits,AND/OR logic gates,and entropy-driven circuits,and respectively demonstrated its significant superiority compared to the original method.We believe that the proposed toehold lock has greatly optimized the efficiency of DNA strand displacement-based networks,and we anticipate that the tool will be widely used in multiple fields.
文摘Nucleic acid sensing is a 3 decades old but still challenging area of application for different biological sub-domains,from pathogen detection to single cell transcriptomics analysis.The many applications of nucleic acid detection and identification are mostly carried out by PCR techniques,sequencing,and their derivatives used at large scale.However,these methods’limitations on speed,cost,complexity and specificity have motivated the development of innovative detection methods among which nucleic acid biosensing technologies seem promising.Toehold switches are a particular class of RNA sensing devices relying on a conformational switch of secondary structure induced by the pairing of the detected trigger RNA with a de novo designed synthetic sensing mRNA molecule.Here we describe a streamlined methodology enabling the development of such a sensor for the RNA-mediated detection of an endangered plant species in a cell-free reaction system.We applied this methodology to help identify the rosewood Dalbergia maritima,a highly trafficked wood,whose protection is limited by the capacity of the authorities to distinguish protected logs from other unprotected but related species.The streamlined pipeline presented in this work is a versatile framework enabling cheap and rapid development of new sensors for custom RNA detection.
基金We would like to thank the National Natural Science Foundation of China （Nos. 91427304, 21434007, 21574122, 51573175, and 21404098）, the National Basic Research Program of China （No. 2012CB821500）, and the Fundamental Research Funds for the Central Universities （WK3450000002 and WK2060200017） for their financial support.
文摘Background： The DNA strand displacement reaction, which uses flexible and programmable DNA molecules as reaction components, is the basis of dynamic DNA nanotechnology, and has been widely used in the design of complex autonomous behaviors. Results： In this review, we first briefly introduce the concept of toehold-mediated strand displacement reaction and its kinetics regulation in pure solution. Thereafter, we review the recent progresses in DNA complex circuit, the assembly of AuNPs driven by DNA molecular machines, and the detection of single nucleotide polymorphism （SNP） using DNA toehold exchange probes in pure solution and in interface state. Lastly, the applications of toehold-mediated strand displacement in the genetic regulation and silencing through combining gene circuit with RNA interference systems are reviewed. Conclusions： The toehold-mediated strand displacement reaction makes DNA an excellent material for the fabrication of molecular machines and complex circuit, and may potentially be used in the disease diagnosis and the regulation of gene silencing in the near future.
文摘Cervical cancer is a global public health subject as it affects women in the reproductive ages,and accounts for the second largest burden among cancer patients worldwide with an unforgiving 50%mortality rate.Relatively scant awareness and limited access to effective diagnosis have led to this enormous disease burden,calling for point-of-care,minimally invasive diagnosis methods.Here,an end-to-end quantitative unified pipeline for diagnosis has been developed,beginning with identification of optimal biomarkers,concurrent design of toehold switch sensors,and finally simulation of the designed diagnostic circuits to assess performance.Using miRNA expression data in the public domain,we identified miR-21-5p and miR-20a-5p as blood-based miRNA biomarkers specific to early-stage cervical cancer employing a multi-tier algorithmic screening.Synthetic riboregulators called toehold switches specific to the biomarker panel were then designed.To predict the dynamic range of toehold switches for use in genetic circuits as biosensors,we used a generic grammar of these switches,and built a neural network model of dynamic range using thermodynamic features derived from mRNA secondary structure and interaction.Second-generation toehold switches were used to overcome the design challenges associated with miRNA biomarkers.The resultant model yielded an adj.R^(2)~0.71,outperforming earlier models of toehold-switch dynamic range.Reaction kinetics modelling was performed to predict the sensitivity of the second-generation toehold switches to the miRNA biomarkers.Simulations showed a linear response between 10 nM and 100 nM before saturation.Our study demonstrates an end-to-end computational workflow for the efficient design of genetic circuits geared towards the effective detection of unique genomic/nucleic-acid signatures.The approach has the potential to replace iterative experimental trial and error,and focus time,money,and efforts.All software including the toehold grammar parser,neural network model and reaction kinetics simulation are available as open-source software(https://github.com/SASTRA-iGEM2019)under GNU GPLv3 licence.
基金supported by National Natural Science Foundation of China(22004010)the Chongqing Science and Technology Commission of China(cstc2019jcyj-msxmX0196)+1 种基金the Science and Technology Research Program of Chongqing Municipal Education Commission(KJQN201901135)the Scientific Research Foundation of Chongqing University of Technology(W.Zhou)
文摘We describe here a target recycling transcription of lighting-up aptamer strategy for detecting ATP in human serums in a label-free means with high sensitivity.ATP molecules specifically recognize the binding aptamer and result in the structure switching of the DNA assembly probes to imitate the target ATP molecule recycling cycles through the toehold-mediated strand displacement reaction,which causes the formation of many dsDNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The organic dye,malachite green,then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals,which can sensitively detect ATP within a dynamic range from 10 to 500 nM in a label-free way.The sensing approach shows a detection limit of 7.3 nM and also has an excellent selectivity for ATP analogue molecules.In addition,this method can detect ATP molecules in diluted human serum samples sensitively,which proves the promising potential to diagnose ATP-related diseases.
